I. Clinical and Genomic Studies of Uveal Coloboma Since initiating this research, I have recruited and examined over 110 famlies where at least one member is affected by uveal coloboma. All probands and their first degree relatives (when available) have complete ophthalmic exams. General physical examinations and targeted systemic testing (e.g., renal ultrasounds, echocardiograms) were performed on probands, as needed. Lymphoblastoid cell lines were established on all participants for candidate gene analysis. Since October 2013, we have published our clinical findings on the yield of systemic testing on patients with relatively isolated uveal coloboma (Am J Ophth). Our analysis revealed that the yield on systemic testing in an outpatient ophthalmology practice was low, especially for clinically-actionable findings. We have turned our attention more recently to the recruitment of patients with more unknown syndromic forms of coloboma. In a pilot experiment using custom-capture, high-throughput sequencing, we have evaluated the yield of testing known coloboma genes in patients with relatively non-syndromic presentation. In our survey of 40 families with coloboma, we identified no convincing mutations, indicating that the yield for diagnostic testing in non-syndromic coloboma is low. These data are being prepared for publication. We are evaluating the pathogenicity of mutations in several additional candidate genes captured in our design. We have established a clinical protocol for whole exome/whole genome analysis, which will allow for the identification of novel genes. We have performed exome analysis on the two pedigrees of autosomal dominant coloboma and missing vertebrae, previously reported. Analysis of candidate genes is underway. We also receive a Clinical Center Genomics Opportunity award to exome 25 trios with syndromic forms of coloboma. II. Laboratory Studies of Uveal Coloboma A. Mouse Models of Coloboma. 1. A novel Pax2 mutant mouse model of coloboma. My lab identified and characterized a mouse model of autosomal dominant congenital optic nerve excavation caused by a missense mutation predicted to change a highly-conserved threonine to alanine in the paired domain of the Pax2 gene. Pax2 is dynamically expressed at the closing edges of the optic fissure and homozygous mutation results in uveal coloboma. Details of our characterization of this model and the Pax2 mutation have been published in PLoS Genetics. Since last annual report, my lab attempted to recapitulate the developmental profiling experiments we performed in wild-type mice (Brown, PNAS) in Pax2 mutant embryos (wild-type vs. heterozygous vs. homozygous mutant) across the three developmental time points for optic fissure closure (E10.5-E12.5). The goal is to identify genes downstream of Pax2 function and to assess these as candidates for mutations in humans. Unfortunately, technical issues limited the usefulness of some critical points in the dataset. We are currently repeating these experiments with appropriate modifications. 2. The RICO Mouse Model of Coloboma The RICO mouse arose from the random insertion of a transgene (NSE-VEGF) on chromosome 13 of C57BL/6 mice. Since the time of the last report, we have identified the junctional fragments of the insertion using whole genome sequencing. We have identified that transgene insertion (30 copies) was associated with an inversion, three duplications and a deletion in a gene desert. We have confirmed the inversion with FISH. We are currently working on an improved method of genotyping embryos and evaluating the effect of the RICO mutant on the expression of genes in the region. B. Identification of coloboma candidate genes by molecular characterization of gene expression during optic fissure closure. 1. Zfp503 and Zfp703 Our previous work, published in PNAS, identified two zinc-finger motif-containing genes, Zfp703 and Zfp503 to be important in regulating optic fissure closure in zebrafish. We have created knockout mice for both Zfp703 and Zfp503 and documented germline transmission, homologous recombination and documented late embryonic lethality for homozygous mutants in both cases. Since the last annual report, we have identified a colobomatous phenotype in both homozygous knockout mice and are currently characterizing the mechanism. In addition, we have made a more careful study of the zfp703 zebrafish morphant phenotype. We have identified that morphant fish have several important phenotypes such as cystic kidneys and abnormalities in heart development. As such, this model likely represents a syndromic form of coloboma. We are screening for mutations in patients and using our zebrafish model to better explore the mechanism of these findings. 2. FAT protocadherins Another gene family that was suggested by our laser-capture screen was the FAT protocadherins. AAs previously described, we have found that Fat1 and Fat4 are the members of this family that are most highly-expressed during embryonic eye development and that homozygous knockout of Fat1--but not Fat4--results in coloboma. We have shown that the coloboma in Fat1-/- mice is not the result of a global patterning defect and that the eyes of these embryos are approximately normal size until the time of optic fissure closure. The rate of cell division in the developing optic cup is mildly elevated compared to wild-type and there is no obvious change in the rate of cell death. Real-time PCR of embryos on a panel of genes has revealed RPE-specific changes in several important cell adhesion molecules and signaling molecules. We are currently extending these studies using a zebrafish model (which also shows coloboma) to determine the precise molecular mechanism behind FAT1-mediated coloboma. 3. Aldh7a1 We recently published (PLoS ONE, Babcock) data on the role of aldehyde dehydrogenase 7a1 in optic fissure closure and fin development in a zebrafish model. Knockdown of aldh7a1 results misregulation of Zfp703 (which can partially rescue the morphant phenotype) and reduced cell division in the optic cup. 4. Prohibitins We have also evaluated the role of prohibitins in optic fissure closure in a zebrafish model of coloboma. Our developmental profiling suggested the PHB1 was differentially regulated in mouse during optic fissure closure and PHB2 was shown by another group to interact with Zfp703. We have shown that knockdown of either phb1 or phb2 in zebrafish results in coloboma. We are currently exploring the molecular mechanisms of these findings.